Size‐Modulated Mesoderm‐Endoderm Divergence and Myocardial Cavitation in Micropatterned Cardioids

The human heart, originating from the splanchnic mesoderm, is the first functional organ to develop, co-evolving with the foregut endoderm through reciprocal signaling. Previously, cardioid models offered new insights on cardiovascular cell lineages and tissue morphogenesis during heart development, while mesoderm-endoderm crosstalk remain incompletely understood. Here, we integrated micropatterned cardioids, CRISPR-engineered reporter hiPSCs, deep-tissue imaging, and single-cell RNA sequencing (scRNA-seq) to explore synergistic mesoderm-endoderm co-development. scRNA-seq with PHATE trajectory mapping reconstructed lineage bifurcations of mesoderm-heart and endoderm-foregut lineages, identifying key cell types in cardiac and hepatic development. Ligand-receptor interaction analysis highlighted mesodermal cells enriched in non-canonical WNT, NRG, and TGF-β signaling, while endodermal cells exhibited VEGF and Hedgehog activity. We found that micropattern sizes influenced cellular composition, cardioid cavitation, contractile functions, and mesoderm-endoderm signaling crosstalk. The cardioids generated from 600 µm diameter circle patterns showed larger cavity formation resembling early heart chamber formation. Our findings establish micropatterned cardioids as a model for mesoderm-endoderm co-development, enhancing our understanding of heart-foregut synergy during early embryogenesis.